In the initial funding period, we made the following observations: 1), the leukemogenic activity of intracellular Notch11 (ICN1) correlates with its ability to activate the transcription factor CSL; to recruit co-activators such as mastermind-like1 (MAML1); drive T cell development; and to inhibit B cell development; 2) blockade of Notch signaling at the level of the plasma membrane or the ICN1/MAML/CSL nuclear complex suppresses the growth of Notch1 leukemia cell lines, suggesting a role for Notch1 in leukemia cell growth and survival; and 3), constitutively active Notch2 and Notch3 also cause T-ALL in our murine model, whereas Notch4 does not. We now propose to build on these findings to clarify ICN structure/leukemia relationships, identify target genes downstream of ICN that are important in Notch leukemia induction and maintenance, and establish the role of co-activator recruitment in leukemogenesis and lymphoid development. Based on our success in suppressing the growth of ICN1-induced T-ALLs with MAML1 peptides, we will conduct a high-throughput screen for small molecule inhibitors of the ICN1/CSL/MAML1 complex. Together, these studies will provide a deeper understanding of Notch-induced leukemia and move us closer to novel therapeutic interventions in this disease. The specific aims of the proposal are: 1. To determine the structural requirements for transformation of T cell progenitors by ICNs 1.1 To Identify the ICN2 and ICN3 domains required for induction of T-ALL 1.2 To reconstitute the ICN4-transforming activity with domain swaps 2. To determine how ICN signals maintain Notch leukemia cell growth 2.1. To determine the domains of ICNs that are required for Notch leukemia cell growth 2.2. To determine the Notch-induced gene expression program that maintains Notch leukemia cell growth 2.3. To identify proteins/signals that are necessary/sufficient for Notch leukemia cell growth 3. To determine the importance of co-activator recruitment in T cell development and transformation 3.1. To identify domains of MAML1 that are essential for function in normal and transformed lymphoid cells 3.2. To identify and characterize small molecule inhibitors of CSL/ICN/MAML1 function